PROJECT SUMMARY Despite aggressive treatment with high-dose inhaled corticosteroids plus bronchodilators, approximately 5-10% of people with asthma (15-30 million) experience severe and life-threatening symptoms of bronchoconstriction. Nearly one-half of these severe asthma patients are also ?sensitized? (i.e. have IgE mediated allergy) to common molds such as Aspergillus fumigatus (Af). This condition has now been recognized as a distinct entity and designated fungal asthma (FA). Unfortunately, current therapies for FA?antifungals and/or omalizumab (anti- IgE)?have not proven to be efficacious in the long-term. By studying Af-induced bronchoconstriction in mice and people with asthma, our team has discovered a novel, direct, and druggable target in FA?the Af allergen-derived serine protease, alkaline protease 1 (Alp1). Alp1 promotes the defining symptom of FA?bronchoconstriction?by inducing airway smooth muscle (ASM) contraction, through mechanisms that appear to be independent of allergic inflammation. In this application, we will pursue the hypothesis that inhibitors of Alp1-induced human ASM contraction can be discovered by high throughput screening (HTS). In aim 1, we will use HTS to identify small molecular inhibitors of Alp1 protease. In aim 2, we will examine the effects of FA pathobiology on the efficacy of Alp1-inhibitor therapy. In aim 3, we will determine mechanism and examine significance for Alp1-inhibitor therapy. We expect our approach to identify novel anti-FA drug candidates, and in doing so, offer substantial insight into both ASM-intrinsic and allergen protease dependent mechanisms of bronchoconstriction.